QUANTITATIVE REAL-TIME PCR BASED DETECTION OF SHRIMP IN COMPLEX FOOD MATRICES  

Anne C. Eischeid
U.S. FDA,
Center for Food Safety and Applied Nutrition,
College Park, MD, 20740,
Anne.Eischeid@fda.hhs.gov

Shrimp-allergic individuals can react to minute quantities of shrimp contained in a relatively large quantity of food, so detection methods must be highly sensitive and specific.  Real-time PCR meets these requirements.  Though shrimp allergens are proteins-and PCR detects DNA-PCR is appropriate for detection of shrimp because the shrimp meat that usually contaminates foods contains both DNA and protein.  Furthermore, well-designed PCR assays distinguish shrimp from other crustacean shellfish allergens, such as crab and lobster, while protein-based ELISA assays do not.  The focus of this talk will be the development and evaluation of a method for real-time PCR-based detection of shrimp in complex food matrices.  Single laboratory evaluation has demonstrated that the method is highly specific for shrimp and performs well in a variety of food matrices subject to various forms of cooking.  This work indicates that real-time PCR is a valuable tool for detection of trace levels of shrimp in foods.  

Shrimp meat was spiked into various foods at 0.1, 1, 10, 100, 1000, 104, and 105 parts per million (ppm).  Samples were homogenized in detergent buffer and DNA was extracted using spin columns.  Real time PCR was carried out using primers and probes designed specifically for this work to target shrimp mitochondrial 12S and 16S genes. CT values from real time PCR were used to plot linear standard curves and their slopes were used to calculate reaction efficiency.  Data are shown using the 12S gene target for shrimp spiked into clam chowder (Fig. 1).  The assay was linear over 8 orders of magnitude (R2 = 0.99) and a reaction efficiency within the ideal range of 100±10%.  Similar results were obtained for both gene targets using other foods and cooking conditions. Cross reactivity tests for real-time PCR were carried out using 250 pg pure target DNA per reaction.  Relative signal strengths indicate that the assay yields robust detection of shrimp and minimal cross-reactivity with other crustaceans (Table 1).